Allan Okey Discusses Applying Microarray Techniques to Mechanistic Toxicology
Emerging Research Front Commentary, December 2010
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Research in several laboratories established that the AHR
is required in order for TCDD to exert its toxic effects. The main
evidence for the essential role of the AHR in dioxin toxicity is that
mice in which the AHR has been knocked out are highly resistant to all
major forms of dioxin toxicity. Given that the AHR is essential to
dioxin toxicity in mice, we reasoned that "candidate genes" that were
revealed in our studies in rats should be regulated by the AHR.
Therefore we wanted to determine whether our candidate genes required the AHR in order to respond to TCDD. Because rat embryonic stem cells had not yet been developed and because targeted gene disruption in rats was not feasible at the time of our research, we used gene expression arrays in AHR-knockout mice to determine which genes are AHR-dependent for their response to TCDD. As a bonus in this research, we also were able to identify the battery of genes that is regulated by the AHR in the absence of any exogenous chemical.
There were no particular challenges or setbacks in this project other than the routine requirements for high-quality laboratory work in obtaining and processing the samples along with the extensive bioinformatics expertise required to analyze the array data.
Where do you see your research leading in the
future?
We continue to extend our search for the genes that are central to dioxin toxicity and also to map AHR-regulated genes and gene networks across species and tissues. It is now clear that gene regulation by the AHR is a physiological phenomenon which is beneficial to the organism when exerted at a controlled level. Therefore determining which gene pathways confer beneficial responses and which pathways lead to toxic responses is of high interest both scientifically and for practical risk assessment.
To identify genes involved in dioxin toxicity we compare phenotypic responses to TCDD in animals that have varied AHR genotypes. For example, Raimo Pohjanvirta succeeded in creating transgenic mice in which the native mouse AHR has been replaced by AHR alleles from either dioxin-sensitive rats or dioxin-resistant rats. Prof. Pohjanvirta's model confirms that the "insertion-variant" AHR obtained from dioxin-resistant rat strains confers a very high degree of resistance to dioxin toxicity in transgenic mice that express this allele (Pohjanvirta Toxicol. Appl. Pharmacol. 236:166-82, 2009).
"Our research used gene expression arrays to interrogate a large fraction of the mouse transcriptome in a single experiment. Our paper is an early example of applying microarray techniques to mechanistic toxicology."
Currently we are conducting gene expression-array experiments in mice carrying variant rat AHR transgenes to determine which of the genes that respond to TCDD are associated with hepatic toxicity and lethality.
Our mapping of AHR-regulated genes across species and tissues reveals that there appears to be a "core battery" of genes that respond to AHR ligands across a wide range of animal species and cell types. "Core" genes include genes that encode CYP1 enzymes that are robustly responsive to AHR agonists and were the first genes discovered to be under AHR control. However, outside this "core" of responsive genes, each species and each cell type seems to possess its own unique repertoire of AHR-regulated genes.
For example, when we compared (Figure 1) the response to TCDD in mouse liver with that in rat liver, 278 genes responded in mouse and 200 genes in rat (Boutros et al., BMC Genomics 9:419 doi:10.1186/1471-2164-9-419, 2008). However, only 33 genes responded in both mouse and rat, indicating wide phylogenetic divergence in AHR regulation, even between these two widely-studied rodent species. Clearly, such profound species differences complicate the issue of which species is "best" as a model for dioxin risk assessment when we attempt to determine the extent to which dioxins are harmful to human health.
AHR-regulated responses to TCDD also can differ widely between tissues within the same animal species. In mice we found that only 17 genes were significantly affected by TCDD in kidney whereas in livers from the same animals 297 genes responded to TCDD in an AHR-dependent fashion (Boutros et al., Toxicol. Sci. 112:245-56, 2009).
It is a challenge to identify genes that respond in a species-selective or cell-selective manner and even more of a challenge to unravel the complex mechanisms that lead to this selectivity. However, the full function of the AHR in "normal biology" as well as the ability of dioxins to selectively target different species and different cell types will not be understood until we have a broader mapping of responsive gene batteries across species and across cell types.
Do you foresee any social or political
implications for your research?
Risk assessment for dioxins in relation to human health has been difficult and controversial because large gaps remain in our knowledge of mechanisms of toxicity as well as understanding why there are striking differences among laboratory species in susceptibility to toxic effects of dioxin-like chemicals. Our research in rodent models is intended to help close these gaps.
In the longer term, studies on the relationship between AHR structure and dioxin toxicity may reveal subgroups of people who are genetically either more prone or less prone than the majority of population to manifest some health impacts of dioxins and, perhaps, other xenobiotic chemicals that act through the AHR.
Our work was supported by grants from the Canadian Institutes for Health Research and the Academy of Finland in the expectation that this research would foster both a better fundamental understanding of AHR biology as well as provide information relevant and useful to practical issues in environmental toxicology.
Submitted by Allan Okey on behalf of Nathalie Tijet, Paul Boutros, Ivy
Moffat, Jouko Tuomisto, and Raimo Pohjanvirta.
Allan B. Okey, Ph.D.
Professor Emeritus
Department of Pharmacology & Toxicology
Faculty of Medicine
University of Toronto
Toronto, Ontario, Canada
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KEYWORDS: 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN TCDD; AH RECEPTOR; MICE LACKING; EXPRESSION; LIVER; INDUCTION; DATABASE; CELLS; IDENTIFICATION; TRANSCRIPTION.